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79/2016 TEXTE

Harmonization of environmental exposure assessment for veterinary pharmaceuticals and biocides: Literature review of studies on occurrence and transformation of veterinary pharmaceuticals and biocides in manure

TEXTE 79/2016

Environmental Research of the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety

Project No. (FKZ) 3712 65 420 Report No. (UBA-FB) 002336/2,ENG

Harmonization of environmental exposure assessment for veterinary pharmaceuticals and biocides: Literature review of studies on occurrence and transformation of veterinary pharmaceuticals and biocides in manure

by

Prof. Dr. Rolf-Alexander Düring, Manuel Wohde Justus Liebig University, Institute of Soil Science and Soil Conservation, Giessen, Germany

Thomas Junker ECT Oekotoxikologie GmbH, Flörsheim, Germany

Dr. Dieter Hennecke, Dr. Monika Herrchen Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany

Prof. Dr. Sören Thiele-Bruhn University Trier, Soil Science Department, Faculty VI, Trier, Germany

On behalf of the German Federal Environment Agency

Imprint

Publisher: Umweltbundesamt Wörlitzer Platz 1 06844 Dessau-Roßlau Tel: +49 340-2103-0 Fax: +49 340-2103-2285 [email protected] Internet: www.umweltbundesamt.de

/umweltbundesamt.de /umweltbundesamt

Study performed by: Justus-Liebig-Universität Giessen, Institut für Bodenkunde und Bodenerhaltung (IFZ) Heinrich-Buff-Ring 26-32 35392 Giessen ECT Oekotoxikologie GmbH Böttgerstraße 2-4 65439 Flörsheim Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie (IME) Auf dem Aberg 1 57392 Schmallenberg Universität Trier Abteilung Bodenkunde, FB VI 54286 Trier Study completed in: March 2016 Edited by: Section IV 2.2 Pharmaceuticals, Washing and Cleaning Agents and Nanomaterials; Dr. Silvia Berkner, Dr. Sabine Konradi Publication as pdf: http://www.umweltbundesamt.de/publikationen ISSN 1862-4804 Dessau-Roßlau, November 2016 The Project underlying this report was supported with funding from the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear safety under project number FKZ 3712 65 420. The responsibility for the content of this publication lies with the author(s).

mailto:[email protected]

http://www.umweltbundesamt.de/

Transformation of veterinary pharmaceuticals and biocides in (liquid) manure

4

Kurzbeschreibung

Die Ausbringung von Veterinärpharmaka und Bioziden mit Gülle auf landwirtschaftlich genutzte Flä-chen stellt einen sehr wichtigen Eintragspfad dieser Stoffgruppen in die Umwelt dar. In dieser Litera-turstudie werden die öffentlich zugänglichen Transformationsstudien von Tierarzneimitteln und Bio-ziden mit Gülle zusammengefasst. Die 34 gefundenen Studien wurden bezüglich des Transformations-verhaltens der getesteten Substanzen, bezüglich der Herkunft und der Spezifikationen der verwende-ten Gülle, bezüglich des experimentellen Setups und bezüglich der gefundenen Ergebnisse ausgewer-tet. Die in den Studien beschriebene Testdauer lag zwischen 2 und 374 Tagen, die Testtemperatur zwi-schen 5 °C und 55 °C. Als Grundaussage aus den 34 Studien kann die sehr große Abhängigkeit des Transformationsverhaltens von der Temperatur, dem Redoxpotential, der Trockenmasse und vieler weiterer Parameter festgehalten werden. Darüber hinaus wurden fehlende Informationen und Para-meter in den gesammelten Studien kritisch herausgearbeitet. Leider wurde zum Beispiel nur in sechs Studien ein Redoxpotential der Gülle bestimmt. Zusätzlich fehlt häufig eine grundsätzliche Charakte-risierung der Matrix Gülle über die Parameter Trockensubstanzgehalt, pH-Wert und total organic car-bon (TOC).

In einem weiteren Teil der Literaturarbeit wurden öffentlich zugängliche Monitoring-Daten gesammelt und ausgewertet. Es wurden folgende Parameter tabellarisch zusammengefasst: Die Herkunft der Gülle, minimal und maximal gefundene Konzentrationen von Tierarzneimittel- und Biozidwirkstoffen, der Anteil an Positivfunden und der Trockensubstanzgehalt der untersuchten Gülle. Insgesamt wurden 39 verschiedene aktive Substanzen und 11 Metabolite und Transformationsprodukte gefunden. Es wurden insgesamt 27 Publikationen ausgewertet, innerhalb derer zusammengenommen 1568 Gülle-Proben analysiert wurden. Innerhalb der einzelnen Publikationen wurde hauptsächlich nach den Wirkstoffklassen der Sulfonamide, der Tetrazykline und der Fluorchinolone gesucht. Einen non-target Ansatz gab es in keiner der Publikationen. Einzelne Wirkstoffe, dieser Substanzklassen wurden in ein-zelnen Studien mit mehr als 100 untersuchten Proben in mehr als 50 % der Fälle gefunden. Dies gilt für Sulfadimidin, Chlortetracyclin, Oxytetracyclin und Tetracyclin.

Es kann festgehalten werden, dass eine standardisierte Herangehensweise sowohl für Transforma-tions- als auch für Monitoring-Studien von Vorteil wäre. Auf diese Weise könnte die Vergleichbarkeit und der wissenschaftliche sowie regulatorische Wert solcher Studien enorm gesteigert werden.


5

Abstract

The spread of veterinary medicinal products (VMPs) and biocides onto agriculturally used areas repre-sents a very important path of entry into the environment for these product groups. Within this litera-ture study public available transformation studies with liquid manure are summarized. Transfor-mation studies were evaluated considering the transformation fate of tested substances, the origin and characteristics of used manure, the experimental setup, the measured parameters and the main out-come of the studies. Test duration throughout the studies ranges from 2 to 374 days and study temper-ature ranges from 5 °C to 55 °C. As main topics within the 34 found transformation studies the high dependency of transformation on temperature, redox potential, dry matter content and many other parameters is reported. It was further critically analyzed which basic information and parameters were neglected. Unfortunately only six publications give information on the redox potential of the manure. Further, the characterization of the matrix in many cases is inadequate due to missing parameters such as dry matter content, pH, and TOC.

Additionally, public available monitoring data of VMPs in manure were collected and evaluated re-garding the origin and characteristics of the manure, the minimum and maximum found concentra-tions, and percentage of identified compounds. Within the 27 found publications, 1568 manure sam-ples were analyzed and 39 different active substances for VMPs and 11 metabolites and transformation products of VMPs could be found in manure. Mainly, the samples were analyzed for sulfonamides, tetracyclines and fluorquinolones. In no case a non-target approach was used. Single active substances were found in some studies with more than 100 analyzed samples in more than 50 % of the analyzed manure samples. This is the case for sulfadimidine, chlortetracycline, oxytetracycline, and tetracy-cline.

It can be concluded that for both transformation studies and monitoring studies a standardized guid-ance would be beneficial for their applicability in regulatory contexts and also enhance the scientific outcome of these studies.


6

Table of contents

Kurzbeschreibung ..................................................................................................................... 4

Abstract .................................................................................................................................... 5

List of figures ............................................................................................................................ 7

List of tables ............................................................................................................................. 7

List of abbreviations .................................................................................................................. 8

Zusammenfassung .................................................................................................................. 10

Summary ................................................................................................................................ 11

1 Introduction: Literature research ...................................................................................... 12

2 Methodology .................................................................................................................. 13

3 Occurrence of veterinary medicines and biocides in manure ............................................... 16

4 Transformation of veterinary pharmaceuticals and biocides in liquid manure ...................... 23

4.1 Citation map ..................................................................................................... 24

4.2 Studied substance classes ................................................................................. 26

4.3 Aerobic vs. anaerobic conditions ........................................................................ 26

4.4 Matrix characteristics and sorption to suspended solids ...................................... 28

4.5 Biotic vs abiotic transformation .......................................................................... 28

4.6 Metabolites and transformation products ............................................................ 29

4.7 Chemical analysis .............................................................................................. 33

4.8 Methane production and microbial activity .......................................................... 33

4.9 Study temperature ............................................................................................. 34

4.10 Source of manure .............................................................................................. 39

4.11 Experimental setup ............................................................................................ 40

4.11.1 N2 flow-through system .................................................................................. 40

4.11.2 Batch assays (no N2 flow-through) .................................................................. 41

4.11.3 Anaerobic sequencing batch reactor (ASBR) .................................................... 44

4.11.4 Realistic outdoor conditions ........................................................................... 45

4.12 Parameters ....................................................................................................... 45

4.13 Conclusions ...................................................................................................... 46

5 References ..................................................................................................................... 47


7

List of figures

Figure 1: Literature selection process steps .......................................... 15

Figure 2 Origin of the 34 transformation studies .................................. 24

Figure 3 Citation Map (created via www.mapequation.org) .................... 25

Figure 4 Investigated substance classes in transformation studies ........ 26

Figure 5 Schematic diagram of microcosm system used for controlling redox potential and pH in Ali and Hernandez (2013) ................ 41

Figure 6 Setup for transformation studies presented by Kühne & Agthe et al. (2000) ............................................................................. 42

Figure 7 Setup for transformation studies presented by Shi and Liang et al. (2011) ............................................................................. 43

List of tables

Table 1: List of categorized keywords .................................................. 13

Table 2: List of requested relevant authorities and organizations .......... 14

Table 3 Sulfonamides and its metabolites found in manure (dw: dry weight, ww: wet weight, ns: not specified) .............................. 17

Table 4 Tetracyclines and its metabolites found in manure (dw: dry weight, ww: wet weight, ns: not specified) .............................. 19

Table 5 Fluorchinolones found in manure (dw: dry weight, ww: wet weight, ns: not specified) ....................................................... 21

Table 6 Other veterinary medicines and its metabolites found in manure (dw: dry weight, ww: wet weight, ns: not specified) .................. 22

Table 7 Studies on transformation of VMPs and biocides in liquid manure and similar matrices (excrements, biosolids, etc, as specified in the second last column) under aerobic and anaerobic conditions. (n.d.: not determined or not defined) ...................................... 30

Table 8 Studies on transformation of VMPs and biocides in liquid manure under aerobic and anaerobic conditions. ................................ 35


8

List of abbreviations

ASBR anaerobic sequencing batch reactor

BOD biological oxygen demand

CAFO Concentrated Animal Feeding Operation

COD chemical oxygen demand

CTC chlortetracycline

CYN cyanamide

DAD diode array detector

DIF difloxacin

DIN german institute for standardization (german: Deutsches Institut für Normung e.V.)

DT50 time needed for disappearance of 50 % of the parent compound, disappearance time

DT90 time needed for disappearance of 90 % of the parent compound, disappearance time

dw dry weight

ECTC 4-epi-chlortetracycline

EDTA ethylenediaminetetraacetic acid

ELISA enzyme-linked immunosorbent assay

EMA European Medicines Agency

ERY erythromycin

EU European Union

F+E research and development (german: Forschung und Entwicklung)

FEN fenbendazole

FKZ project no. (german: Forschungskennzahl)

FLU flubendazole

GC gas chromatography

HPLC high performance liquid chromatography

HR-MS high resolution - mass spectrometry

ICTC iso-chlortetracycline

IMZ imazalil

ISO International Organization for Standardization

LC liquid chromatography

LIN lincomycin

LOD limit of detection

LSC liquid scintillation counting

MON monensin

MS mass spectrometry

n.d. not determined or not defined


9

NER non-extractable residues

ns not specified

OECD Organisation for Economic Cooperation and Development

OTC oxytetracycline

RTLC radio thin layer chromatography

SCP sulfachloropyridazine

SDZ sulfadiazine

SMZ sulfamethoxazole

SPN spectinomycin

TMP trimethoprim

TOC total organic carbon

TP transformation product

TS total solids

TYL tylosin

UV ultraviolet

VDI Association of German Engineers (german: Verein Deutscher Ingenieure )

VFA volatile fatty acid

VICH Veterinary International Conference on Harmonization

VMP veterinary medicinal product

VSS volatile suspended solids

ww wet weight


10

Zusammenfassung

Die Ausbringung von Veterinärpharmaka und Bioziden mit Gülle auf landwirtschaftlich genutzte Flä-chen stellt einen sehr wichtigen Eintragspfad dieser Stoffgruppen in die Umwelt dar. Aktuelle Bewer-tungsleitfäden (zum Beispiel: „Guideline on determining the fate of veterinary medicinal products in manure“ (EMA/CVMP/ERA/430327/2009) (EMA 2011) sehen aus diesem Grund auch experimentelle Untersuchungen zur Transformation dieser Substanzen in Gülle vor.

Allerdings beinhalten die Dokumente bisher lediglich nur grundlegende regulatorische Vorgaben. Eine experimentelle Prüfrichtlinie zur Durchführung von Studien zum Abbauverhalten von Veterinär-pharmaka und Bioziden in Gülle liegt jedoch weder auf EU- noch auf OECD-Ebene vor. Um eine ein-heitliche Bewertung von Studien im Zulassungsverfahren zu gewährleisten, wird jedoch ein harmoni-siertes, international akzeptiertes und validiertes Testverfahren benötigt. Vor diesem Hintergrund wurde im Rahmen des F+E-Vorhabens „Entwicklung einer Testvorschrift zum Abbauverhalten von Ve-terinärpharmaka und Bioziden in Gülle“ (FKZ 3710 67 422) (Hennecke et al. 2015) ein Entwurf für eine experimentelle Richtlinie erarbeitet.

In dieser Literaturstudie werden die öffentlich zugänglichen Transformationsstudien von Tierarznei-mitteln und Bioziden mit Gülle zusammengefasst. Die 34 gefundenen Studien wurden bezüglich des Transformationsverhaltens der getesteten Substanzen, bezüglich der Herkunft und der Spezifikatio-nen der verwendeten Gülle, bezüglich des experimentellen Setups und bezüglich der gefundenen Er-gebnisse ausgewertet. Die in den Studien beschriebene Testdauer lag zwischen 2 und 374 Tagen, die Testtemperatur zwischen 5 °C und 55 °C. Als Grundaussage aus den 34 Studien kann die sehr große Abhängigkeit des Transformationsverhaltens von der Temperatur, dem Redoxpotential, der Trocken-masse und vieler weiterer Parameter festgehalten werden. Darüber hinaus wurden fehlende Informa-tionen und Parameter in den gesammelten Studien kritisch herausgearbeitet. Leider wurde zum Bei-spiel nur in sechs Studien ein Redoxpotential der Gülle bestimmt. Zusätzlich fehlt häufig eine grund-sätzliche Charakterisierung der Matrix Gülle über die Parameter Trockensubstanzgehalt, pH-Wert und total organic carbon (TOC).

In einem weiteren Teil der Literaturarbeit wurden öffentlich zugängliche Monitoring-Daten gesammelt und ausgewertet. Es wurden folgende Parameter tabellarisch zusammengefasst: Die Herkunft der Gülle, minimal und maximal gefundene Konzentrationen von Tierarzneimittel- und Biozidwirkstoffen, der Anteil an Positivfunden und der Trockensubstanzgehalt der untersuchten Gülle. Insgesamt wurden 39 verschiedene aktive Substanzen und 11 Metabolite und Transformationsprodukte gefunden. Es wurden insgesamt 27 Publikationen ausgewertet, innerhalb derer zusammengenommen 1568 Gülle-Proben analysiert wurden. Innerhalb der einzelnen Publikationen wurde hauptsächlich nach den Wirkstoffklassen der Sulfonamide, der Tetrazykline und der Fluorchinolone gesucht. Einen non-target Ansatz gab es in keiner der Publikationen. Einzelne Wirkstoffe, dieser Substanzklassen wurden in ein-zelnen Studien mit mehr als 100 untersuchten Proben in mehr als 50 % der Fälle gefunden. Dies gilt für Sulfadimidin, Chlortetracyclin, Oxytetracyclin und Tetracyclin.

Es kann festgehalten werden, dass eine standardisierte Herangehensweise sowohl für Transforma-tions- als auch für Monitoring-Studien von Vorteil wäre. Auf diese Weise könnte die Vergleichbarkeit und der wissenschaftliche sowie regulatorische Wert solcher Studien enorm gesteigert werden.


11

Summary

The spread of veterinary medicinal products (VMPs) and biocides onto agriculturally used areas repre-sents a very important path of entry into the environment for these product groups. For this reason, current guidance (e.g. „Guideline on determining the fate of veterinary medicinal products in ma-nure“(EMA/CVMP/ERA/430327/2009) (EMA 2011) stipulates experimental studies on transformation of veterinary medicinal products (VMPs) and biocides in manure.

Though, the documents only contain basic regulatory requirements, whereas an experimental test guideline is still missing, both on EU and OECD level. To allow for a consistent assessment of studies within the registration process, a harmonized internationally accepted and validated test method is needed. A draft test guideline was developed within a previous R&D-Project “Development of test guid-ance for transformation of veterinary pharmaceuticals and biocides in liquid manure” (FKZ 3710 67 422) (Hennecke et al. 2015).

Within this literature study public available transformation studies with liquid manure are summa-rized. Transformation studies were evaluated considering the transformation fate of tested substances, the origin and characteristics of used manure, the experimental setup, the measured parameters and the main outcome of the studies. Test duration throughout the studies ranges from 2 to 374 days and study temperature ranges from 5 °C to 55 °C. As main topics within the 34 found transformation studies the high dependency of transformation on temperature, redox potential, dry matter content and many other parameters is reported. It was further critically analyzed which basic information and parameters were neglected. Unfortunately only six publications give information on the redox potential of the ma-nure. Further, the characterization of the matrix in many cases is inadequate due to missing parameters such as dry matter content, pH, and TOC.

Additionally, public available monitoring data of VMPs in manure were collected and evaluated re-garding the origin and characteristics of the manure, the minimum and maximum found concentra-tions, and percentage of identified compounds. Within the 27 found publications, 1568 manure sam-ples were analyzed and 39 different active substances for VMPs and 11 metabolites and transformation products of VMPs could be found in manure. Mainly, the samples were analyzed for sulfonamides, tetracyclines and fluorquinolones. In no case a non-target approach was used. Single active substances were found in some studies with more than 100 analyzed samples in more than 50 % of the analyzed manure samples. This is the case for sulfadimidine, chlortetracycline, oxytetracycline, and tetracy-cline.

It can be concluded that for both transformation studies and monitoring studies a standardized guid-ance would be beneficial for their applicability in regulatory contexts and also enhance the scientific outcome of these studies.


12

1 Introduction: Literature research

Veterinary medicinal products (VMPs) are excreted by the treated animals as parent and metabolized compounds. The excrements from stabled animals in Europe and North America are collected and stored mainly as liquid or solid manure before they are used as fertilizers on arable land and grassland. Biocides, which are used for disinfection of the stable, end up in the stored animal excrements. Via manure application in agriculture, veterinary medicines and biocides are released to the environment and affect soil and water quality.

Depending on boundary conditions such as storage temperature, dry matter content, feeding of the animals and availability of electron acceptors, the pharmaceuticals and biocides can be further trans-formed in the liquid manure. Besides transformation other processes such as volatilization, sorption, and the formation of non-extractable residues (NER) can occur and contribute to the dissipation of the active agents.

Transformation products are also capable to persist in environmental matrices and can be ecotoxic. For tetracyclines the transformation products like epimers, isomers and anhydro-compounds have been detected. Metabolites of sulfadiazine showed transformation back to the parent compound. Transformation processes are influenced by the composition of matrix, temperature, pH value as well as aerobic or anaerobic conditions. Compounds could adsorb to the matrix depending on its sorption capacity. The higher the dry matter content in liquid manure, the higher is the number of sorption sites.

Generally, transformation under aerobic conditions occurs faster than transformation under anaerobic conditions. Also high temperatures promote the degradation of compounds in liquid manure. During manure storage in manure tanks, which is mostly applied in Europe, the storage conditions are anaer-obic. In North America manure is stored in lagoons because of the large amounts of manure that accu-mulates in large-scale Concentrated Animal Feeding Operations (CAFOs). The outdoor lagoon storage is distinguished by more aerobic conditions on the large lagoon water surface but also by anaerobic conditions in deeper layers. Composting the separated manure under aerobic conditions is the favored treatment of manure in Asia. Concluding, the transformation process of compounds is affected in-tensely by the storage practice of manure.

There is increasing research activity regarding the transformation of single substances under labora-tory conditions. Current guidance, e.g. „Guideline on determining the fate of veterinary medicinal products in manure“(EMA 2011), takes transformation of VMPs and biocides in manure into account. However, currently, there is no standardized experimental test protocol available to examine the trans-formation of veterinary medicinal products (VMPs) and biocides in liquid manure. The EMA guideline on transformation in manure (EMA 2011) contains basic regulatory requirements. To allow for a con-sistent assessment of studies within regulatory frameworks, a harmonized internationally accepted and validated test method is needed.

This literature study provides a survey on studies on (bio-) transformation processes in manure and on monitoring data of VMPs and biocides in manure. The objective of this review was to follow the ques-tions which compounds are investigated, which methods and analytical techniques are used and which factors have been identified affecting the (bio-) transformation process in liquid manure. In ad-dition, this report compiles recent monitoring data on VMP and biocide residues in manure samples.


13

2 Methodology

For this literature review the data bases “ISI Web of Knowledge” and “Google Scholar” were used. Cat-egorized search items are given in Table 1. Boolean search for the most relevant two keywords, which were then combined with the following categories could limit the results to a manageable amount of citations.

Table 1: List of categorized keywords

1. 2. 3. 4. 5. 6.

manure transfor-mation

veterinary medicine biocide florfenicol

slurry metabolism drug pesticide tetracycline feces catabolism pharmaceuti-

cal disinfectant sulfonamide

faeces anabolism antibiotic lagoon degradation antiparasitic

decomposi-tion

dissipation fate reaction conversion management

International publications from the year 2000 to date were considered. In addition, cross references within the original publications were verified. Additionally, relevant authorities and organizations were asked for reports on related topics, see Table 2.

Furthermore, proven experts in this field were contacted in Canada and South Korea to assess specific local situations abroad:

Dr. Edward Topp Agriculture and Agri-Food Canada 1391 Sandford Street London, ON N5V 4T3 Canada

Dr. Jin-Wook Kwon Ministry for Food, Agriculture Forestry and Fisheries 620-2 Amnam-dong Seo-gu, Busan Rep. of Korea


14

Table 2: List of requested relevant authorities and organizations

Ministerium für Energiewende, Umwelt, Landwirtschaft und ländliche Räume des Landes Schleswig-Holstein

Landesamt für Natur, Umwelt und Verbraucherschutz NRW (LANUV), Recklinghausen

Bayerisches Staatsministerium für Ernährung, Landwirtschaft und Forsten

Bayerische Landesanstalt für Landwirtschaft (LfL)

Bayerisches Staatsministerium für Umwelt und Gesundheit, München

Institut für Agrarökologie, Ökologischen Landbau und Bodenschutz (IAB), Freising

Thüringer Ministerium für Landwirtschaft, Forsten, Umwelt und Natur-schutz; Abteilung Landwirtschaft, Markt, Ernährung

Thüringer Landesanstalt für Landwirtschaft (TLL), Jena, Abteilung Tierpro-duktion

Landwirtschaftliches Zentrum für Rinderhaltung, Grünlandwirtschaft, Milchwirtschaft, Wild und Fischerei Baden Württemberg (LAZBW); Aulen-dorf

Sächsische Landesanstalt für Landwirtschaft

Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern

Hessisches Landesamt für Umwelt und Geologie

Biogas e.V.

Biogas Union

Citations were collected and entered into the software package “Citavi” (Swiss Academic Software, for Microsoft Windows). The established data base comprises literature which, in the broadest sense, deals with environmental burden due to active agents originated from husbandry. The results of this re-search were structured according to the following categories:

• Transformation of veterinary medicinal products and biocides in manure • Transformation in soil and in soil/manure mixtures • Sorption • Fate in the aquatic system • Stable Manure / Composting • Treatment • Antibiotic resistance genes • Monitoring • Biogas/ methane (antibiotics) • Copper • Metabolism in the animal


15

• Plant uptake • Regulatory aspects • International agricultural studies • Miscellaneous

From 648 entered records, 27 were found to measure or monitor the occurrence of VMPs in liquid ma-nure. Found substances, estimated concentrations, origin of the manure and further parameters were listed. From 648 records 34 publications were selected which deal explicitly with transformation of veterinary medicinal products and biocides in pig and cattle manure. These citations were systemati-cally transferred to an Excel data base which considered specific parameters such as investigated com-pounds and substance amounts, characterization of matrices, aerobic/anaerobic conditions, transfor-mation products, methodology and chemical analytics of the studies.

Figure 1: Literature selection process steps

Literature database (648 citations)

Evaluation databses

27 citations for monitoring studies

34 citations for transformation studies

Analysis of literature


16

3 Occurrence of veterinary medicines and biocides in manure

The monitoring data tables (Tables 3-6) summarize the results of 27 different publications measuring veterinary medicinal products in manure from 2001 until today. Manure was analyzed in North Amer-ica (Canada), in Europe (Austria, Czech Republic, Denmark, Germany, Italy, Switzerland) and in Asia (China, Japan). The gathered results give a good general survey on the prevalent occurrence of phar-maceuticals in manure. Although it should be kept in mind, that the data do only cover certain loca-tions, it can be assumed, that wherever veterinary medicinal products are used, portions of these will be found in the manure.

In some studies, a lot of samples were taken covering a large number of manures - up to 380 samples in Harms et al. (2005) - and in other studies only individual manures were sampled with the back-ground knowledge of the previous medication. Within 19 publications only pig manure was analyzed, whereas 3 publications worked with cattle manure and 4 publications worked with pig, cattle or poul-try manure. One publication did not specify the origin of the analyzed manure.

Manure and liquid manure samples with different dry matter content were considered in this literature study (range: 0.2 - 44.4 %). 16 of 27 Studies unfortunately did not specify dry matter content as a basic parameter. 12 of 27 studies reported concentrations of substances in manure in mg/kg dry weight (dw), 10 of 27 studies worked with mg/kg wet weight (ww) and 5 studies did not specify (ns) whether they calculated concentrations on the basis of dry or wet weight. Because of this, it is difficult to compare the found concentrations of the single active substances. The lowest values are found at the µg/kg order of magnitude - often restricted by the limit of detection (LOD) of the analytical method. For future studies it would be highly desirable to get at least all the information provided: Dry weight and wet weight concentrations of analyzed compound and dry matter content.

Among all reviewed literature, 39 different active substances of VMPs were found in manure. Moreo-ver, 11 metabolites and transformation products of these active substances were also identified. For this, 1568 manure samples were analyzed within the 27 publications. Mainly, the samples were ana-lyzed for sulfonamides, tetracyclines and fluorquinolones. None of the studies worked with a non-tar-get approach. By far the most frequently found single active substances are sulfadimidine (599 posi-tive), tetracycline (575 positive) and chlortetracycline (457 positive). There are 6 publications each of which analyzed more than 100 manure samples. All these come from Chinese or German institutes. Those active substances with the highest percentage of positive findings (> 50 %) within these 6 pub-lications are chlortetracycline, oxytetracycline, tetracycline and sulfadimidine.

The 15 highest concentrations are found in pig manure from Germany or China. The highest concen-tration was 1420.76 mg/kg (dw) of enrofloxacin found in poultry manure from China, followed by 764.407 mg/kg (dw) chlortetracycline in pig manure from China and 330.7 mg/kg (ww) in pig manure from Germany. Further, very high values are found for other sulfonamides, tetracyclines and fluorchi-nolones.


17

Table 3 Sulfonamides and its metabolites found in manure (dw: dry weight, ww: wet weight, ns: not specified)

Substance Reference Matrix Origin Min Max Unit Dry matter content, comments or

quotation n n positive

% posi-tive

Sulfachloropyridazine Hu et al. 2010 pig and poultry manure China 0.340 3.660 mg/kg (dw) "liquid swine manure" (ns) 6 2 33 Zhao et al. 2010 pig, cattle and poultry manure China 0.090 3.510 mg/kg (dw) (ns) 143 7 5

Sulfadiazine Engels 2004 pig manure Germany (NI) 0.700 235.100 mg/kg (ww) 0.5 - 16.8 % (mean 5 %) 344 100 29 Hamscher et al. 2005 pig manure Germany 3.500 11.300 mg/kg (dw) 9.6 - 9.8 % 3 2 67 Harms et al. 2005 pig manure Germany (BY) 0.100 5.000 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 19 5 Hu et al. 2010 pig and poultry manure China 0.160 0.780 mg/kg (dw) "liquid swine manure" (ns) 6 2 33

Jacobsen and Halling Sørensen

2006 pig manure Denmark 0.630 2.100 mg/kg (dw) 2.8 - 13.4 % 6 2 33

Pfeifer et al. 2002 (ns) Germany 0.011 0.080 mg/kg (ns) "liquid manure" (ns) 4 2 50 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.650 mg/kg (dw) liquid and stable manure (ns) 34 5 15 Winckler et al. 2004 pig manure Germany 0.700 35.300 mg/kg (ww) 0.7 - 16.11 % 176 86 49 Zhao et al. 2010 pig, cattle and poultry manure China 0.020 3.120 mg/kg (dw) (ns) 143 14 10

4-Hydroxy-Sulfadiazine Ratsak et al. 2013 pig and cattle manure Germany (NW) - 9.050 mg/kg (dw) liquid and stable manure (ns) 34 8 24

N4-Acetyl-Sulfadiazine Harms et al. 2005 pig manure Germany (BY) - - not quantified 0.2 - 17.3 % (mean 3.7 %) 380 19 5 Pfeifer et al. 2002 (ns) Germany 0.010 0.270 mg/kg (ns) "liquid manure" (ns) 4 2 50 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.150 mg/kg (dw) liquid and stable manure (ns) 34 6 18

Sulfadimethoxine Harms et al. 2005 pig manure Germany (BY) 0.050 0.600 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 5 1 Pan et al. 2011 pig manure China 0.120 1.255 mg/kg (dw) (ns) 126 3 2

Sulfadimidine Martìnez-Carballo et al. 2007 pig manure Austria - < 20 mg/kg (dw) "liquid manure" (ns) 30 18 60 Aust et al. 2008 cattle manure Canada - 9.990 mg/kg (dw) 24.4 - 44.4 % (mean 37 %) 6 4 67 Burkhardt et al. 2005 pig manure Switzerland - 14.400 mg/L (ww) "in the supernatant" (water phase) (ns) 1 1 100 Christian et al. 2003 pig manure Germany 1.000 1.100 mg/kg (ww) (ns) 2 2 100 Christian et al. 2003 cattle manure Germany < 0.1 < 0.1 mg/kg (ww) (ns) 2 2 100 Engels 2004 pig manure Germany (NI) 0.700 167.000 mg/kg (ww) 0.5 - 16.8 % (mean 5 %) 344 183 53 Hamscher et al. 2005 pig manure Germany - 7.200 mg/kg (dw) 9.6 - 9.8 % 3 1 33 Pan et al. 2011 pig manure China 0.011 28.700 mg/kg (dw) (ns) 126 65 52 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 7.040 mg/kg (dw) liquid and stable manure (ns) 34 6 18 Sattelberger et al. 2005 pig manure Germany 0.130 20.000 mg/kg (dw) 1.2 - 28 % 30 18 60 Weiß 2008 pig manure Germany (BY) 0.140 1.700 mg/L (ww) 3 - 2 % 8 8 100 Winckler et al. 2004 pig manure Germany 0.700 167.000 mg/kg (ww) 0.7 - 16.13 % 176 85 48 Zhao et al. 2010 pig, cattle and poultry manure China 0.060 6.040 mg/kg (dw) (ns) 143 17 12 Haller et al. 2002 pig and cattle manure Switzerland 0.130 8.700 mg/kg (ww) 1.1 - 3.7 % 6 6 100 Harms et al. 2005 pig manure Germany (BY) 0.050 38.000 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 181 48


18

Pfeifer et al. 2002 (ns) Germany 0.011 0.062 mg/kg (ns) "liquid manure" (ns) 4 2 50

N4-Acetyl-Sulfadimi-dine

Haller et al. 2002 pig and cattle manure Switzerland < 0.1 2.600 mg/kg (ww) 1.1 - 3.7 % 6 5 83

Harms et al. 2005 pig manure Germany (BY) 0.050 27.000 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 117 31 Weiß 2008 pig manure Germany (BY) 0.120 1.000 mg/L (ww) 2 - 2 % 8 8 100

Sulfadoxine Hu et al. 2010 pig and poultry manure China 0.350 0.710 mg/kg (dw) "liquid swine manure" (ns) 6 3 50



Sulfaguanidine Zhao et al. 2010 pig, cattle and poultry manure China 0.010 1.550 mg/kg (dw) (ns) 143 27 19

Sulfamerazine Harms et al. 2005 pig manure Germany (BY) 0.700 0.900 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 7 2 Zhao et al. 2010 pig, cattle and poultry manure China 0.090 0.660 mg/kg (dw) (ns) 143 6 4

N4-Acetyl-Sulfamera-zine

Harms et al. 2005 pig manure Germany (BY) - - not quantified 0.2 - 17.3 % (mean 3.7 %) 380 5 1

Sulfamethizole Pan et al. 2011 pig manure China 0.052 2.422 mg/kg (dw) (ns) 126 35 28

Sulfamethoxazole Harms et al. 2005 pig manure Germany (BY) 0.050 0.050 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 3 1 Hu et al. 2010 pig and poultry manure China 0.340 1.290 mg/kg (dw) "liquid swine manure" (ns) 6 2 33 Motoyama et al. 2011 pig manure Japan 0.002 0.035 mg/kg (ns) (ns) 5 4 80 Motoyama et al. 2011 cattle manure after fermentation Japan - 0.010 mg/kg (ns) (ns) 8 1 13 Pan et al. 2011 pig manure China 0.137 0.639 mg/kg (dw) (ns) 126 6 5 Sattelberger et al. 2005 pig manure Germany < 0.1 2.400 mg/kg (dw) 1.2 - 28 % 30 2 7 Zhao et al. 2010 pig, cattle and poultry manure China 0.120 2.800 mg/kg (dw) (ns) 143 7 5

Sulfamethoxypyridazine Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.020 mg/kg (dw) liquid and stable manure (ns) 34 4 12

Sulfamonomethoxine Motoyama et al. 2011 pig manure Japan - 0.210 mg/kg (ns) (ns) 5 1 20 Motoyama et al. 2011 cattle manure after fermentation Japan - 0.022 mg/kg (ns) (ns) 8 1 13 Zhao et al. 2010 pig, cattle and poultry manure China 0.060 4.080 mg/kg (dw) (ns) 143 39 27

Sulfanilamide Zhao et al. 2010 pig, cattle and poultry manure China 0.020 1.590 mg/kg (dw) (ns) 143 5 3

Sulfaquinoxaline Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.670 mg/kg (dw) liquid and stable manure (ns) 34 3 9

Sulfathiazole Haller et al. 2002 pig and cattle manure Switzerland 0.100 12.400 mg/kg (ww) 1.1 - 3.7 % 6 4 67 Harms et al. 2005 pig manure Germany (BY) 0.050 0.100 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 5 1 Pan et al. 2011 pig manure China 0.312 mg/kg (dw) (ns) 126 1 1


19

Table 4 Tetracyclines and its metabolites found in manure (dw: dry weight, ww: wet weight, ns: not specified)



% posi-tive

Chlortetracycline Martìnez-Carballo et al. 2007 pig manure Austria 0.100 46.000 mg/kg (dw) "liquid manure" (ns) 30 17 57 Engels 2004 pig manure Germany (NI) 1.100 330.700 mg/kg (ww) 0.5 - 16.8 % (mean 5 %) 344 44 13 Hamscher et al. 2002 pig manure Germany 0.090 0.100 mg/kg (ww) (ns) 2 2 100 Hamscher et al. 2005 pig manure Germany 0.900 1.000 mg/kg (dw) 9.6 - 9.8 % 3 2 67 Harms et al. 2005 pig manure Germany (BY) 0.100 50.800 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 140 37 Hu et al. 2010 pig and poultry manure China 0.150 14.700 mg/kg (dw) "liquid swine manure" (ns) 6 4 67



Motoyama et al. 2011 pig manure Japan 0.240 0.280 mg/kg (ns) (ns) 5 2 40 Motoyama et al. 2011 cattle manure after fermentation Japan - 0.001 mg/kg (ns) (ns) 8 1 13 Pan et al. 2011 pig manure China 0.053 764.407 mg/kg (dw) (ns) 126 122 97 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 3.600 mg/kg (dw) liquid and stable manure (ns) 34 7 21 Sattelberger et al. 2005 pig manure Germany 0.100 46.000 mg/kg (dw) 1.2 - 28 % 30 17 57 Tylová et al. 2010 pig manure Czech Republic - 5.880 mg/kg (ns) "Liquid hog manure" (ns) 5 1 20 Weiß 2008 pig manure Germany (BY) 0.600 2.000 mg/L (ww) 1 - 2 % 3 3 100 Winckler et al. 2004 pig manure Germany 1.100 25.700 mg/kg (ww) 0.7 - 16.1 % 176 18 10 Zhao et al. 2010 pig, cattle and poultry manure China 0.160 27.590 mg/kg (dw) (ns) 143 72 50

Epi-Chlortetracycline Jacobsen and Halling Sørensen


Doxycycline Harms et al. 2005 pig manure Germany (BY) 0.100 0.700 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 5 1



Tylová et al. 2010 pig manure Czech Republic - 0.990 mg/kg (ns) "Liquid hog manure" (ns) 5 1 20 Zhao et al. 2010 pig, cattle and poultry manure China 0.230 13.500 mg/kg (dw) (ns) 143 21 15

Metacycline Zhao et al. 2010 pig, cattle and poultry manure China 0.140 5.860 mg/kg (dw) (ns) 143 50 35

Oxytetracycline Martìnez-Carballo et al. 2007 pig manure Austria 0.290 29.000 mg/kg (dw) "liquid manure" (ns) 30 22 73 De Liguoro et al. 2003 cattle manure Italy - 19.000 mg/kg (ns) "heap" (ns) 1 1 100 Engels 2004 pig manure Germany (NI) 1.600 136.200 mg/kg (ww) 0.5 - 16.8 % (mean 5 %) 344 10 3 Harms et al. 2005 pig manure Germany (BY) 0.100 0.900 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 16 4




20

Motoyama et al. 2011 pig manure Japan - 0.013 mg/kg (ns) (ns) 5 1 20 Motoyama et al. 2011 cattle manure after fermentation Japan - 0.001 mg/kg (ns) (ns) 8 1 13 Pan et al. 2011 pig manure China 0.044 172.874 mg/kg (dw) (ns) 126 114 90 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 1.490 mg/kg (dw) liquid and stable manure (ns) 34 5 15 Sattelberger et al. 2005 pig manure Germany 0.210 29.000 mg/kg (dw) 1.2 - 28 % 30 22 73 Winckler et al. 2004 pig manure Germany 1.600 136.200 mg/kg (ww) 0.7 - 16.9 % 176 9 5 Zhao et al. 2010 pig, cattle and poultry manure China 0.150 59.590 mg/kg (dw) (ns) 143 50 35 Karcı and Balcıoğlu 2009 cattle manure Turkey - 0.060 mg/kg (ns) (ns) 1 1 100

Epi-Oxytetracycline Jacobsen and Halling Sørensen


Tetracycline Martìnez-Carballo et al. 2007 pig manure Austria 0.360 23.000 mg/kg (dw) "liquid manure" (ns) 30 22 73 Hamscher et al. 2002 pig manure Germany 3.200 4.000 mg/kg (ww) (ns) 2 2 100 Hamscher et al. 2005 pig manure Germany 14.100 41.200 mg/kg (dw) 9.6 - 9.8 % 3 3 100 Harms et al. 2005 pig manure Germany (BY) 0.100 46.000 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 111 29 Hu et al. 2010 pig and poultry manure China 0.180 0.840 mg/kg (dw) "liquid swine manure" (ns) 6 4 67



Motoyama et al. 2011 pig manure Japan 0.005 0.015 mg/kg (ns) (ns) 5 3 60 Motoyama et al. 2011 cattle manure after fermentation Japan - 0.001 mg/kg (ns) (ns) 8 2 25 Pan et al. 2011 pig manure China 0.037 19.417 mg/kg (dw) (ns) 126 107 85 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 2.450 mg/kg (dw) liquid and stable manure (ns) 34 12 35 Sattelberger et al. 2005 pig manure Germany 0.360 23.000 mg/kg (dw) 1.2 - 28 % 30 22 73 Winckler and Grafe 2001 pig manure Germany (NW) 0.600 66.000 mg/L (ww) "pig slurry" (ns) 181 43 24 Winckler et al. 2004 pig manure Germany 0.900 43.100 mg/kg (ww) 0.7 - 16.8 % 176 87 49 Engels 2004 pig manure Germany (NI) 0.700 45.700 mg/kg (ww) 0.5 - 16.8 % (mean 5 %) 344 152 44

Epi-Tetracycline Jacobsen and Halling Sørensen



21

Table 5 Fluorchinolones found in manure (dw: dry weight, ww: wet weight, ns: not specified)



% posi-tive

Ciprofloxacin Motoyama et al. 2011 pig manure Japan - 0.006 mg/kg (ns) (ns) 5 1 20 Motoyama et al. 2011 cattle manure after fermentation Japan 0.002 0.012 mg/kg (ns) (ns) 8 4 50 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.070 mg/kg (dw) liquid and stable manure (ns) 34 3 9 Sattelberger et al. 2005 pig manure Germany 0.180 0.620 mg/kg (dw) 1.2 - 28 % 30 4 13 Weiß 2008 pig manure Germany (BY) 0.005 0.028 mg/L (ww) 5 - 2 % 5 5 100 Zhao et al. 2010 pig, cattle and poultry manure China 0.490 45.590 mg/kg (dw) (ns) 143 44 31

Danofloxacin Ratsak et al. 2013 pig and cattle manure Germany (NW) 0.050 mg/kg (dw) liquid and stable manure (ns) 34 1 3 Zhao et al. 2010 pig, cattle and poultry manure China 0.080 3.060 mg/kg (dw) (ns) 143 39 27

Difloxacin Zhao et al. 2010 pig, cattle and poultry manure China 0.410 12.380 mg/kg (dw) (ns) 143 8 6

Enrofloxacin Martìnez-Carballo et al. 2007 pig manure Austria 0.130 0.750 mg/kg (dw) "liquid manure" (ns) - - - Ratsak et al. 2013 pig and cattle manure Germany (NW) 0.550 mg/kg (dw) liquid and stable manure (ns) 34 5 15 Sattelberger et al. 2005 pig manure Germany 0.130 0.750 mg/kg (dw) 1.2 - 28 % 30 5 17 Weiß 2008 pig manure Germany (BY) 0.050 0.116 mg/L (ww) 6 - 2 % 5 5 100 Zhao et al. 2010 pig, cattle and poultry manure China 0.330 1420.760 mg/kg (dw) (ns) 143 67 47

Fleroxacin Zhao et al. 2010 pig, cattle and poultry manure China 0.760 99.430 mg/kg (dw) (ns) 143 35 24

Levofloxacin Motoyama et al. 2011 pig manure Japan - 0.003 mg/kg (ns) (ns) 5 1 20 Motoyama et al. 2011 cattle manure after fermentation Japan 0.001 0.002 mg/kg (ns) (ns) 8 2 25

Lomefloxacin Zhao et al. 2010 pig, cattle and poultry manure China 0.610 44.160 mg/kg (dw) (ns) 143 45 31

Marbofloxacin Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.050 mg/kg (dw) liquid and stable manure (ns) 34 3 9

Norfloxacin Zhao et al. 2010 pig, cattle and poultry manure China 0.560 225.450 mg/kg (dw) (ns) 143 37 26

Ofloxacin Hu et al. 2010 pig and poultry manure China 0.450 3.870 mg/kg (dw) "liquid swine manure" (ns) 6 2 33

Orbifloxacin Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.020 mg/kg (dw) liquid and stable manure (ns) 34 1 3

Sarafloxacin Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.060 mg/kg (dw) liquid and stable manure (ns) 34 1 3


22

Table 6 Other veterinary medicines and its metabolites found in manure (dw: dry weight, ww: wet weight, ns: not specified)



% posi-tive

Flubendazole Weiß 2008 pig manure Germany (BY) 0.020 0.056 mg/L (ww) 8 - 2 % 7 7 100

Amino-Flubendazole Weiß 2008 pig manure Germany (BY) 0.032 0.110 mg/L (ww) 7 - 2 % 7 7 100

Hydroxy-Flubendazole Weiß 2008 pig manure Germany (BY) 0.018 0.075 mg/L (ww) 9 - 2 % 7 7 100

Lincomycin Kuchta and Cessna 2009 pig manure Canada 2.520 9.780 mg/L (ww) mean 2.4 % 5 5 100

Salinomycin Schlüsener et al. 2003 pig manure Germany - 0.011 mg/kg (ns) 5 % 4 1 25

Spectinomycin Kuchta and Cessna 2009 pig manure Canada 0.173 0.686 mg/L (ww) mean 2.4 % 5 5 100

Tiamulin Harms et al. 2005 pig manure Germany (BY) - 0.500 mg/kg (ww) 0.2 - 17.3 % (mean 3.7 %) 380 1 < 1 Pan et al. 2011 pig manure China 0.076 0.169 mg/kg (dw) (ns) 126 6 5 Schlüsener et al. 2003 pig manure Germany - 0.043 mg/kg (ns) 5 % 4 1 25

Toltrazuril Olsen et al. 2012 pig manure Denmark - 0.114 mg/kg (dw) "manure from a slurry storage tank" (ns) 1 1 100

Toltrazuril sulfone Olsen et al. 2012 pig manure Denmark - 0.085 mg/kg (dw) "manure from a slurry storage tank" (ns) 1 1 100

Toltrazuril sulfoxide Olsen et al. 2012 pig manure Denmark - 0.007 mg/kg (dw) "manure from a slurry storage tank" (ns) 1 1 100

Trimethoprim Haller et al. 2002 pig and cattle manure Switzerland < 0.1 < 0.1 mg/kg (ww) 1.1 - 3.7 % 6 1 17 Ratsak et al. 2013 pig and cattle manure Germany (NW) - 0.050 mg/kg (dw) liquid and stable manure (ns) 34 1 3

Tylosin De Liguoro et al. 2003 cattle manure Italy - < 0.25 mg/kg (ns) "heap" (ns) 1 1 100 Solliec et al. 2014 pig manure Canada 0.030 0.543 mg/kg (dw) (ns) - - - Weiß 2008 pig manure Germany (BY) 0.130 0.320 mg/L (ww) 4 - 2 % 8 8 100


23

4 Transformation of veterinary pharmaceuticals and biocides in liq-uid manure

The focus of the literature search was on transformation studies with liquid manure and manure from lagoons. Liquid manure is the substrate found in a manure tank which consists of urine, feces, some-times bedding material and water from cleaning the stables. It is important to note the difference to dung or excrements, which are distinguished from manure by being directly excreted and not collected and stored in some form for longer time periods under which anaerobic conditions develop (Weinfurt-ner 2011). In this review also studies using excrements and related matrices were included to get a comprehensive picture of available methods.

In literature many different studies using mixtures of soil and manure or test systems containing addi-tionally plants can be found. These were not considered for the survey.

Studies on solid manure (mainly conducted at Asian institutions) are not considered in this review. The composition of this material is considerably more variable than the composition of liquid manure matrix which results in i.e. wide ranges of oxygen availability. Compared to solid manure, liquid ma-nure exhibits a more homogeneous composition. This type of manure was considered primarily, as it has been found to be the predominant type of manure in the EU countries and North America (Wein-furtner 2011).

Out of a total of 648 publications a limited number of 34 relevant studies was selected. These studies together with information on their experimental design are compiled in Table 7 and Table 8. On the whole, there is only scarce data on transformation of veterinary medicinal products and especially on transformation of biocides.

Generally, the research on transformation of pharmaceuticals in manure is focused on North America, Europe, and Asia (Figure 2). However, there is an increasing publication rate worldwide which reflects the impact and necessity of this research field


24

Figure 2 Origin of the 34 transformation studies

4.1 Citation map

The following citation map (Figure 3) provides an impression on the interconnection of the au-thors/working groups by generating a network and their respective impact in this field of research. Each node represents one publication. The darker and the bigger the node, the more often the publica-tion is cited. The arrows show who cites whom, their thickness correlates with the citation flow indi-cating established thematic clusters. Only three publications are completely left out citing each other, owed to dealing with hormones and lagoon water. One isolated work of Varel (2002) considers delib-erate application of (natural) biocides to manure. This was to stop microbial activity and prevent “odor emissions” during the storage of manure. This approach is contrary to all other considered publica-tions. One cluster is implied on the right of this network, showing seven publications which used 14C-labeled compounds, all originating from Germany (working groups Kreuzig and Spiteller). Most often cited papers are from Kühne et al. (2000) and Loke et al. (2000). This is partly explainable by the rela-tively early date of publication.


25

Figure 3 Citation Map (created via www.mapequation.org)


26

4.2 Studied substance classes

A compilation of the investigated active substances (on the basis of frequentness of substance classes) is depicted in Figure 4. Equivalent to the application practice in livestock breeding, mainly sulfona-mide and tetracycline antibiotics were considered. There are only a few studies with parasiticides. For biocides only three publications were found (Kreuzig & Schlag et al. 2010, Kreuzig 2010, Varel 2002) Within 2 of 34 studies transformation of excreted hormones was investigated. Although they are not about VMPs, these publications were also considered because they are well documented (e.g. meas-ured redox potential) and conducted similar to the rare transformation studies with VMPs.

Figure 4 Investigated substance classes in transformation studies

4.3 Aerobic vs. anaerobic conditions

It was tried to select studies that were conducted under primarily anaerobic conditions, as stated by the authors or deduced from the given information on the experimental set up. However, also studies are included in the table which used aerobic conditions (e.g. redox potentials above -100 mV, OECD 2002). Various authors assume anaerobic conditions without any further indication. Different termi-nology is used, such as “anaerobic digestion”, “anaerobic conditions”, “methanogenic conditions”, and "anaerobic tightly capped” vessels. Studies with this vague information were considered in this closer examination, otherwise the relevant publications would have been limited to a number of only six studies reporting a redox potential.

Besides using closed laboratory setups, many studies report the use of N2 or He gas to purge the head-spaces of the systems or to purge the used liquids and manures before starting the experiments. Others added reducing agents to the manures to guarantee reducing conditions (e.g. Na2S by Álvarez & Omil et al. (2010) or titanium(III)citrate by Loke & Tjørnelund et al.(2003)). Loke & Tjørnelund et al. (2003)

0

2

4

6

8

10

12

14

16

Tetracyclines Sulfonamides Macrolides Biocides Others


27

further added resazurin as redox indicator. As the test bottles did not show a reddish coloring, they assumed anaerobic conditions. However, it might be difficult to interpret the coloring of this also pH-dependant indicator in deep brown liquid manure. For this, they also monitored methane gas produc-tion as a main indicator for methanogenic – and by this anaerobic - conditions. Varel & Parker et al. (2012) systematically studied methane production of their seed manure before starting transformation studies with this manure, to being able to work with stable methanogenic conditions.

Kuhne et al. (2000) used closed incubation systems to investigate the stability of tetracycline in pig manure. They determined that the DT50 (disappearance time 50 %) for tetracycline in their unventi-lated systems was 9 days, whereas it was 4.5 days when the slurry was ventilated.

Szatmári et al. (2011) compared an anaerobic laboratory study with a field study using manure com-posting. In the laboratory experiment more than 30 % and in the field study about 10 % of the initial doxycycline amount could be detected in manure samples after 16 and 12 weeks of manure ageing period, respectively. The half-life of doxycycline in manure was calculated to be 52.5 days under an-aerobic conditions and 25.7 days under aerobic conditions.

Ali et al. (2013) were the only authors from all included studies who deliberately varied the redox po-tential. They established a set of microcosms with controlled redox potentials (Eh) (-100 mV, 0 mV, +250 mV, and +350 mV) and pH conditions (pH 5.5, 7.0, and 8.5). With increasing Eh - that is with increasing aerobic conditions - they found significantly increased dissipation rates for tylosin and could attribute this to microbial activity by comparison with sodium azide treated samples. Increasing pH resulted in increasing dissipation rates. With the addition of azide a decrease of Eh could be meas-ured. Kolz et al. (2005) worked with redox potentials of slurry between -10 and -80 mV. The addition of azide resulted in a decrease of the redox potential to between -90 and -160 mV.

Although redox potential is not directly a proof of anaerobic conditions, it is a relatively easy to meas-ure parameter to get insight into the system liquid manure. The internationally harmonized guideline concerned with transformation of chemicals in water sediment systems pragmatically sets a maximal upper limit of -100 mV (OECD 2002) for anaerobic conditions. Strictly considering this limit of -100 mV, only three studies meet the requirements for anaerobic conditions (Ali et al. (2013), Lamshöft et al. (2010), Zheng et al. (2013)). It should be considered, that redox potentials measured in real manure tanks are even much below -100 mV (Weinfurtner 2011). Generally, the transformation of VMPs in manure is faster and more complete under aerobic conditions than under anaerobic conditions.


28

4.4 Matrix characteristics and sorption to suspended solids

From the 34 studies, eight used cattle manure which showed dry matter contents from 1.1 up to 13 %. Two studies used both swine and cattle manure. One study relies on a synthetic matrix-water mixture to approximate properties of liquid manure. For the remaining 23 studies, swine manure was the ma-trix exhibiting dry matter contents from 2 up to 15.2%. Again, and similar to the point of varying oxy-gen availability, comparability is complicated due to differing dry matter contents. Kreuzig emphasizes substance specific interactions with the different pig or cattle manure matrices (Kreuzig 2010). He fur-ther mentioned that the dry substance content of manure is one of the most relevant factors affecting the transformation of VMPs and biocides. With a study on the stability of tylosin A in manure Loke et al. could not clarify whether the decrease in the concentration of this compound is caused by sorption, abiotic or biotic chemical degradation (Loke et al. 2000). Similarly, Shi et al. could not explain whether the rapid disappearance of the investigated antibiotics tetracycline and sulfamethoxydiazine could be due to their adsorption onto solid materials or degradation by microorganisms (Shi et al. 2011). In another study Loke et al. (2003) stated that very low free concentrations of oxytetracycline and metab-olites in an anaerobic degradation experiment is due to high amounts of substances being bound to particles in the manure matrix rather than degradation to unknown compounds.

In 15 studies this dry matter content which is strongly influencing sorption of the test substances is not even mentioned, prohibiting a deeper interpretation of the results. As this is a key parameter which impacts the dissipation rate, could be shown by Álvarez et al. (2010), Arikan (2008), Kolz et al. (2005), Kreuzig (2010) and Kuchta and Cessna (2009). These authors investigated explicitly sorption onto solid matter which was already recognized as a crucial parameter by Winckler and Grafe (2001).

4.5 Biotic vs abiotic transformation

In some studies either sterilization with sodium azide or autoclaving allowed to differentiate between abiotic and biotic transformation. Generally, biotic transformation rates are substantially higher than pure abiotic transformation rates. However, the process of formation of non-extractable residues can-not be elucidated by this approach.

Loke & Tjørnelund et al. (2003) worked with autoclaved and non-autoclaved manure to study trans-formation of oxytetracycline (OTC) by measuring the free concentration of the VMP. By this, they did not find a difference between the sterile and the non-sterile setups, due to a fast sorption of OTC to the solid phase. They did not address the question, whether biotic transformation is inhibited by sorption of the substance to suspended solids.

Loke & Tjørnelund et al. (2000) studied transformation of tylosin A but were unfortunately not able to figure out whether the rapid decrease in the concentration of tylosin A is caused by sorption, abiotic or biotic chemical degradation. Ali & Hernandez et al. (2013) later reported among other conclusions, that under aerobic (Eh +350 mV) conditions, microbial degradation was much greater than under an-aerobic conditions compared to abiotic transformation or sorption. Kolz & Douglass et al. (2005) con-cluded that both biodegradation and abiotic degradation occur during transformation of tylosin. How-ever, strong sorption to slurry solids was probably the primary mechanism of tylosin disappearance.


29

Zheng & Bradford et al. (2012) found, that transformation of 17 α-estradiol, 17 β-estradiol, and estrone was mainly dominated by biodegradation than by physical or chemical transformation.

Li et al. found that the combined processes of hydrolysis and biodegradation were responsible for transformation of ceftiofur. The determined hydrolysis and total degradation rate constants in aqueous solutions varied with temperature (Li et al. 2011).

4.6 Metabolites and transformation products

With regard to VMPs it is important to distinguish between metabolites, which may be formed during metabolism in the treated animal, and transformation products, which may be formed from excreted parent and metabolites in the environment.

Transformation products were determined in 22 studies. This implies sophisticated methodology by liquid chromatography coupled to preferably tandem mass spectrometry or high resolution mass spec-trometry (LC-MS/MS or LC-HR-MS). For specific applications, HPLC (high performance liquid chroma-tography) with UV (ultraviolet) detection may be sufficient (Winckler and Grafe 2001). Due to missing standard substances, transformation products are often determined only qualitatively.

Arikan (2008) studied the fate of chlortetracycline (CTC) during anaerobic digestion of manure from medicated calves. The CTC concentration decreased about 75 % and the concentration of the CTC epi-mer, 4-epi-chlortetracycline (ECTC), declined roughly 33 % during the 33 day experiment. The CTC metabolite, iso-chlortetracycline (ICTC), increased 2-fold in concentration. Referring to a higher water solubility, the authors concluded a possible occurrence of metabolites of CTC in water bodies.

Also Mitchell et al. stated that solid and liquid effluents from anaerobic digestion treatment with anti-biotic transformation products could present an environmental concern (Mitchell et al. 2013).

In the study of Heuer et al. (2008) the concentration of Sulfadiazine (SDZ) increased by 42 % during storage of the manure due to deacetylation of the metabolite N-acetyl-SDZ. Basically the same was de-termined by Lamshöft et al. (2010) who alert environmental effects may be underestimated if the par-ent compound alone is considered for environmental risk assessment.


30

Table 7 Studies on transformation of VMPs and biocides in liquid manure and similar matrices (excrements, biosolids, etc, as specified in the second last column) under aerobic and anaerobic conditions. (n.d.: not determined or not defined)

Author Year Substances Substance class TP Initial

concentration DT50

Mineraliza-tion

Manure (type and source)

Dry matter

Ali & Hernandez et al.

2013 Tylosin Macrolide - 160 mg/L n.d. (highly pH and Eh depend-ant)

n.d. cattle (spiked, mixed lagoon sedi-ment)

2.7 %

Álvarez & Omil et al.

2010 Oxytetracycline (OTC), Chlortetracy-cline (CTC)

Tetracycline + 10, 50, 100 mg/L 15.4 -12.0 (OTC), 4.1 - 3.2 (CTC) d

n.d. pig (spiked, tank) -

Angenent & Raskin et al.

2008 Tylosin-A Macrolide + 5.8 mg/L (measured) 2.49 h n.d. pig (spiked, tank/ASBR)

-

Arikan 2008 Chlortetracycline Tetracycline + 1.0 and 5.9 mg/L (EDTA-Buffer Extrac-tion, pH 4)

18 d n.d. pig (medicated, mixed excrements)

5 %

Arikan & Foster et al.

2006 Oxytetracycline Tetracycline + 9.8 mg/L 56 d n.d. cattle (medicated, mixed excrements)

5 %

Blackwell & Noble et al.

2005 Oxytetracycline (OTC), Sulfachloro-pyridazine (SCP)

Tetracycline, Sul-fonamide

- 19.2 (OTC), 26.1 (SCP) mg/L

79 (OTC), 127 (SCP) d

n.d. pig (spiked, tank) 2 %

Cetecioglu & Ince et al.

2013 Tetracycline Tetracycline - Gradient: 1.65, 5.7, 8.5 mg/L

n.d. n.d. synthetic (spiked, ASBR)

-

Grote & Freitag et al.

2004 Chlortetracycline (CTC), Sulfadiazine (SDZ), Trimethoprim (TMP)


+ up to: 87.5 (CTC), 498.9 (SDZ), 15.8 (TMP) mg/kg

n.d. n.d. pig (medicated, ‘bar-rels’)

-

Harms 2006 20 different sub-stances

Tetracycline, Sul-fonamide, and others

- numerous, many not given

n.d. n.d. pig (medicated & spiked, tank)

-

Heuer & Spiteller et al.

2008 Sulfadiazine (14C) Sulfonamide + > 80 mg/kg n.d. (DT50 not reached)

< 1 % pig (medicated, mixed excrements)

6 %


31

Höltge & Kreuzig 2007 Sulfamethoxazole, Acetyl-Sulfamethoxa-zole (each 14C)

Sulfonamide and metabolite

+ 3 mg/kg n.d. ≤ 1 % cattle (spiked, mixed excrements)

13 %

Kolz & Douglass et al.

2005 Tylosin Macrolide + 20 and 195 mg/L DT90: 40 - 500 h n.d. pig (spiked, lagoon water)

1.5, 3.6 %

Kreuzig 2010 Erythromycin (ERY), Sulfamethoxazole (SMZ), Cyanamide (CYN), Imazalil (IMZ), (each 14C)

Macrolide, Sul-fonamide, Bio-cide, Imidazole

- only absolute radio-activity given; 0.1 to 0.2 MBq

n.d. < 0.1 % (ERY, SMZ);

28 % (CYN); n.d. for (IMZ)

pig, cattle (spiked, mixed excrements)

2.5, 5, 10 %

Kreuzig & Höltge 2005 Sulfadiazine (14C) Sulfonamide - 500 µg/kg 17 d 1 % cattle (spiked, mixed excrements)

13 %

Kreuzig & Höltge et al.

2007 Fenbendazole (FEN), Flubendazole (FLU), (each 14C)

Benzimidazole + 200 (FEN), 2500 (FLU) µg/kg

n.d. (DT50 not reached)

< 0.6 % pig (spiked, mixed excrements)

3 – 13 %

Kreuzig & Schlag et al.

2010 Imazalil (14C) Imidazole + 4.3 and 4.5 mg/kg > 177 d 0.1 % pig, cattle (spiked, mixed excrements)

2.5, 5, 10 %

Kuchta & Cessna 2009 Lincomycin (LIN), Spectinomycin (SPN)

Antimicrobial - 38.7 (LIN), 387 (SPN) µg/L

n.d. n.d. pig (spiked, lagoon water)

-

Kühne & Agthe et al.

2000 Tetracycline Tetracycline + 200 mg/L 9 d n.d. pig (spiked, tank) -

Lamshöft & Spitel-ler et al.

2010 Difloxacin (DIF), Sul-fadiazine (SDZ), (each 14C)

Fluoroquinolone, Sulfonamide

+ 17.1 ± 0.4 (DIF), 156.0 ± 4.2 (SDZ) mg/L

n.d. (DT50 not reached)

0.2 % (DIF), 0.5 % (SDZ)

pig (medicated, mixed excrements)

3.3 - 6 %

Li & Katterhenry et al.

2011 Ceftiofur β-Lactam antibi-otic

+ 19.1 µmol/L 1.7 - 41 (highly dependant on T and dilution ratio with water)

n.d. cattle (spiked, ‘wa-ter from farm’)

1.1 %

Loke & Tjørnelund et al.

2003 Oxytetracycline Tetracycline + 2 and 30 mg/L n.d. n.d. pig (spiked, tank) -


2000 Tylosin A Macrolide + 5 mg/L < 2 d n.d. pig (spiked, tank) -


32

Mitchell & Frear et al.

2013 Ampicilin, Florfenicol, Sulfamethazine, Tylo-sin

β-Lactam antibi-otic, Amphenicol, Sulfonamide, Macrolide

+ each 0.001 - 1.0 mM/L

n.d. n.d. cattle (spiked, mixed excrements)

3 - 6 %

Mohring & Ham-scher et al.

2009 8 Sulfonamides Sulfonamide + 2 - 14 mg/kg n.d. n.d. pig (spiked, biogas plant)

15.2 %

Schlüsener & Bester et al.

2006 Erythromycin, Rox-ithromycin, Salinomy-cin, Tiamulin

Macrolide, Iono-phore, Pleuromu-tilin

+ 2 mg/kg 6 d - >180 d n.d.

pig (spiked, tank) -

Shelver & Varel 2012 Chlortetracycline Tetracycline + >100 and >300 ng/L (only given in fig-ures)

> 21 d at 22 °C, < 5 d at 38 °C and 55 °C

n.d. pig (medicated, mixed excrements)

-

Shi & Liang et al. 2011 Tetracycline, Sulfa-methoxydiazine


- each 25 and 50 mg/L < 12 h n.d. pig (spiked, mixed excrements)

10 %

Stone & Spellmann et al.

2009 Chlortetracycline (CTC), Tylosin (TYL)

Tetracycline, Mac-rolide

+ 28 (CTC), 1.1 (TYL) mg/L

n.d. n.d. pig (medicated, ma-nure)

-

Szatmári & Borbély et al.

2011 Doxycycline Tetracycline - 61.57 ± 14.26 mg/kg

53 d n.d. pig (medicated, ma-nure)

-

Varel 2002 Carvacrol, Thymol Terpenoid - each 6.7 - 16.75 mmol/L

n.d. n.d. pig (spiked, mixed excrements)

-

Varel & Parker et al.

2012 Chlortetracycline (CTC), Monensin (MON)

Tetracycline, Ion-ophores

- 5.9 - 8.3 (CTC), 0.3 -0.74 (MON) mg/L

n.d. (DT50 not reached for MON)

n.d. pig, cattle (medi-cated, seed slurry and manure)

4 %

Winckler & Grafe 2001 Tetracycline Tetracycline - 20 and 100 mg/L 55 - 105 d n.d. pig (spiked, tank) -

Zheng & Bradford et al.

2012 17-β-estradiol, 17-α-estradiol, estrone

Hormone + each 5 mg/L n.d. n.d. cattle (spiked, la-goon water)

-

Zheng & Machew-ski et al.

2013 17 α-estradiol-3-sul-fate

Conjugate of a Hormone

+ 5 mg/L 23 - 724 d n.d. cattle (spiked, la-goon water)

1.2 %


33

4.7 Chemical analysis

As already mentioned with regard to the citation map, seven studies used 14C labeled test substances. By this, a mass balance of the experiment considering transformation, mineralization, volatilization, and the formation of non-extractable residues is possible. The methods used are radio thin layer chro-matography (RTLC), oxidizers for solid samples and liquid scintillation counting (LSC). Only Heuer & Spiteller et al. (2008) and Lamshöft & Spiteller et al. (2010) further used LC-MS techniques in combi-nation with radio techniques. An approach that will be inevitable in future studies to gain maximum information out of transformation studies in terms of transformation product identification and quan-tification.

Exemplary, Höltge and Kreuzig investigated the fate of 14C-labeled sulfamethoxazole and acetyl-sulfa-methoxazole in a laboratory experiment (Höltge and Kreuzig 2007). Within an incubation period of 72 d, 1 % at maximum of the initially applied radiotracers was released as 14CO2 while more than 75 % was transferred to non-extractable residues that were operationally defined by an ethyl acetate extrac-tion. In long-term degradability tests, the authors could determine an increase of non-extractable res-idues and 11 % mineralization at maximum.

However, by far most of the studies worked with unlabeled substances and used LC-MS or LC-MS/MS for detection and quantification of the VMPs and biocides and their transformation products (18 pub-lications). Some of them combined UV-Vis / diode array detector (DAD) methods with MS-methods (3 publications). For example Schlüsener and Bester et al. (2006) used HR-MS (high resolution - mass spectrometry) for further salinomycin transformation product identification. Within 7 publications only UV-Vis/DAD-detection methods were used. The only GC method (gas chromatography) was ap-plied by Varel (2002) for the detection of the terpenoids carvacrol and thymol. Varel (2012) applied an ELISA method (enzyme-linked immunosorbent assay) for the detection of chlortetracycline.

4.8 Methane production and microbial activity

The production of methane was considered as ongoing parameter in eight studies. In two studies with tetracyclines, Arikan et al. (2006) and Alvarez et al. (2010) found that methane production was re-duced by 27 % during batch experiments and up to 62 % due to antibiotic dosage, respectively. Stone et al. (2009) found that generation of methane was inhibited by 27.8 % due to the presence of chlor-tetracycline. Dependent on the dosage, Cetecioglu et al. (2013) determined adverse impact of tetracy-cline with a total collapse of the microbial activity and metabolic functions at a concentration of 8.5 mg/L in a synthetic substrate mixture under anaerobic conditions. Shi et al. (2011) found a dosage dependent inhibition on CH4 production and concluded antibiotics appear to inhibit bacterial activity resulting in a delay and overall decline in CH4 production. Among these eight studies, two studies were explicitly concerned with microbiological issues (Stone et al. 2009; Heuer et al. 2008). Varel & Parker et al. (2012) mentioned that a 5 to 6 months adaption period was necessary for acclimatization of mi-croorganisms to monensin and to reduce effects of antimicrobals on methane production. Against that background each future transformation study has to be analyzed critically. Composition of microbial community has a massive effect on transformation rates and routes. Without any further qualitative


34

and quantitative critical analysis of microbiology, it is not possible to produce reliable and reproduci-ble transformation data of VMPs and biocides in liquid manure. From a regulatory point of view, this topic could enable a massive manipulation of transformation data.

4.9 Study temperature

Study temperatures ranged from 5 °C to 55 °C. Few publications study the temperature effect in detail by variation of study temperature.

Harms (2006) examined the stability of pharmaceuticals in manure during storage at 7 °C and found no degradation of chlortetracycline during six months. Sulfadiazine was reduced to 50 % after one week, but the remaining residuals were stable until the end of the trial (32 weeks). The author varied storage temperatures (-20°C, 7°C, room temperature) over a period of 16 weeks. Sulfamerazine, sulfa-methoxypyrazine, sulfaguanidine and sulfisomedine persisted. Sulfamethoxazole was reduced up to 80 % at 7°C as well as at room temperature. The same degradation was reached more quickly with higher temperatures than under cooler conditions of storage. Enrofloxacin and tiamulin were reduced to 20 % at 7°C storage temperature. For tiamulin a reduction of 10 % at 7 °C and 25 % at room temper-ature was observed. Only sulfapyridazine and enrofloxacine showed a small decline at a storage tem-perature of -20 °C.

Li et al. (2011) studied the transformation of ceftiofur at temperatures between 15 °C and 45 °C and found increasing hydrolysis and biodegradation rates with temperature. Increasing study temperature from 35 °C to 45 °C resulted in an increasing relevance of hydrolysis on transformation of ceftiofur, whereas biodegradation remained static.

Similarly, Varel et al. (2012) found principally increasing dissipation rates with increasing tempera-ture from 22°C to 55°C. They studied the effect of anaerobic digestion at different temperatures, among other parameters, on the fate of chlortetracycline (CTC) in swine manure and monensin (MON) in cattle manure. The authors concluded that anaerobic digestion at elevated temperatures may be an effective treatment to reduce CTC but not to reduce MON. Transformation of CTC mainly depends on abiotic transformation. This was also shown by Shelver et al. (2012), who also worked with CTC between 22°C and 55°C

Stone et al. (2009) worked with a temperature gradient to simulate field conditions commonly found in the northern mid-western United States of America. They started with 10°C (0-30 days) and in-creased temperature over time: 12°C (30-46 days), 15°C (46-56 days) and 20°C (56-216 days).

Approximately half of the studies (16 out of 34) took place at ambient temperatures (ranging from 20°C to 25°C). Some other experiments ran at elevated temperatures of 35°C to 40°C which enhances micro-bial activity (Arikan 2008). In general, transformation was found to be dependent on study tempera-ture, with an increase in transformation rate with increasing temperature. Working with temperatures above the microbiological relevant limit of 35-40 °C results in an inhibition of microbial activity and biodegradation processes. In what way this effects transformation processes, mainly depends on the transformation routes for different substances.


35

Table 8 Studies on transformation of VMPs and biocides in liquid manure under aerobic and anaerobic conditions.

Author Year Focus and parameters Setup Amount manure Preconditioning/accli-matization

Repli-cates

Study-T [°C]

Eh [mV]

Study du-ration [d]

Ali & Hernandez et al.

2013 pH and Eh 2.3 L erlenmeyer flask, continuously stirred and

flushed with N2/O2 for different Eh (Fig. 5)

150 g wet lagoon sediment + 1.5 L

0.01 M CaCl2

1 week for stabilization of pH and Eh

1 25 (-100), (0),

(250), (350)

20

Álvarez & Omil et al. 2010 biogas compostion, pressure, sorption

500-mL glass flasks with coiled butyl rubber

stoppers

385 mL + Inoculum (granular biomass from an anaerobic internal circulation

digester)

Basal medium: cys-teine (0.5 g/L), NaHCO3

(5 g/L), pH 7.0–7.2; flushing with N2, 1.2 mL Na2S (20 g/L) (re-

ducing agent)

2 35 - 21

Angenent & Raskin et al.

2008 antibiotic resistance, me-thane production, vola-tile solids removal, VFA

manure taken from ASBR effluent, 5 mL

capped glass serum vi-als

1 mL 249 days of ASBR oper-ation

1 25 - 2

Arikan 2008 sorption, pH, total solids, volatile solids, total alka-

linity, NH4-N, COD

1 L batch laboratory di-gester

800 mL manure + 200 mL inoculum from from a dairy manure digester

n.d. 3 35 - 33

Arikan & Foster et al.

2006 biogas production, total solids, total alkalinity,

total N, total P

1.225 L batch labora-tory digester

1 L manure + 225 mL inoculum from

from a dairy manure digester

n.d. 3 35 - 64

Blackwell & Noble et al.

2005 exposure assessment, organic carbon, dry mat-

ter, available P and N

closed bottle test, tightly capped and

stored without agitation

200 mL n.d. 3 20 - 40

Cetecioglu & Ince et al.

2013 synthetic manure, COD, biogas production

ASBR, concentration in-fluent and effluent,

sludge

1 L 150 days of ASBR oper-ation

1 35 - 155


36

Grote & Freitag et al.

2004 metabolism, transfor-mation

outdoor realistic condi-tions with continuous influent of contami-

nated manure

“barrels” n.d. 1 outdoor - 240 + 210

Harms 2006 transformation n.d. n.d. n.d. n.d. -20, 7, RT - 112, 224

Heuer & Spiteller et al.

2008 bacterial community n.d. n.d. n.d. 1 20 - 172

Höltge & Kreuzig 2007 transformation, NER 300-mL flasks, glass stoppers with inlet and

outlet valves, 14CO2-trap

50 g 7 days 3 20 - 72

Kolz & Douglass et al.

2005 aerobic vs anaerobic, sorption, pH, total solids,

N, TOC, P

amber glass vials with teflon-lined caps, head-

space filled with He

20 mL ‘homogenized stored in glass jars at 4 °C until

use’

3 22 (-10) - (-160)

3

Kreuzig 2010 T,Eh, dry matter, O2, N-to-tal, NH4-N, TOC, BOD

300-mL flasks, glass stoppers with inlet and

outlet valves, 14CO2-Trap

50 g n.d. 2 5, 10, 20 (- 80) 30, 100, 177

Kreuzig & Höltge 2005 transformation, NER 300-mL flasks, glass stoppers with inlet and


50 g n.d. 2 20 - 102

Kreuzig & Höltge et al.

2007 manure-soil mixtures, transformation, NER



50 g n.d. 2 20 - 102

Kreuzig & Schlag et al.

2010 manure-soil mixtures, transformation, NER, bio-

cides



50 g n.d. 2 20 (< 120) 177

Kuchta & Cessna 2009 sorption, liquid-solid dis-tribution after centrifuga-

tion

20-L stainless-steel storage container with

clipdown cover

15.5 L n.d. 2 20 - 160

Kühne & Agthe et al.

2000 transformation vacuum desiccator (Fig. 6)

1 L n.d. 2 RT - 8

Lamshöft & Spitel-ler et al.

2010 T, Eh, dry mass, pH, BOD, COD, total carbon, con-

ductivity

300-mL flasks with 14CO2-Trap

50 g ‘the manure was al-lowed to attain room

temperature’

3 10, 20 (- 280) - (- 329)

150


37

Li & Katterhenry et al.

2011 TOC, conductivity, pH, P,NH4-N, Cl-, Br-, NO3-,

Na, K, Ca, Fe, Mg, Al, Si, Cu, Zn

Amber 250 mL bottles with teflon lined caps

served as reactors

< 250 mL n.d. 3 15, 25, 35, 45

- 72


2003 pH, Eh via indicator, freely dissolved fraction

according to ISO 11734 (ISO 1998), 1 L bottles;

titanium(III)citrate as re-ducing agent

525.0 ml mineral medium, 50.0 ml

manure 100.0 ml stock solu-

tion

< 2 weeks storage at 4 °C

4 21 - 180


2000 transformation, filtered vs non-filtered

according to ISO 11734 (ISO 1998), volumes x

50, 680 mL

680 mL (water with 6,4 % manure)

1 mm sieved, N2 bub-bled through manure, stored at 4 and -20 °C

before usage

4 20 - 7

Mitchell & Frear et al.

2013 pH, CH4, CO2 inhibi-tion,total solids (TS)

and VSS

300 mL glass serum bottles fitted with rub-

ber septum, headspace filled with N2, inoculum

used

200 mL n.d. 3 37 - 40

Mohring & Ham-scher et al.

2009 biogas production, pH 5-L fermentors (Bigatec, Rheinberg, Germany),

German VDI 4630 guide-line, DIN 38414 Part 8, control experiments in

500 mL flasks

1.89 kg manure, 1.89 L water,

0.42 kg inoculum

n.d. 2 37 - 34

Schlüsener & Bester et al.

2006 transformation Erlenmeyer flasks closed with a ferment-

ing tube

100 g n.d. 1 20 - 180

Shelver & Varel 2012 pH, transformation 2 L digester flasks n.d. n.d. 3 22, 38, 55

- 28

Shi & Liang et al. 2011 methane production, pH, total solids

1 L digester with gas ab-sorbing bottle and col-

lector-bottle (Fig. 7)

1 L (including 100 g dry manure, 100 mL

inoculum)

n.d. 3 25 - 20


38

Stone & Spellmann et al.

2009 CH4, CO2, volatile fatty acids, pH, Alkalinity,

COD, VSS, VFA, hydro-gentrophic methano-

gens, aceticlastic meth-anogens

120 mL batch reactors, butyl rubber stoppers, headspace N2 purged

50 g 105 d at 4 °C 3 10-20 (gradient)

- 216

Szatmári & Borbély et al.

2011 transformation 300 ml BOD-bottles as used in closed bottle

tests; referring to VICH/EMA (2010)

< 300 mL n.d. n.d. 20 - 112

Varel 2002 odor, total gas, VFA, L-lactate, pH

1 L Erlenmeyer flasks, N2-gas, rubber stopper

500 mL (faeces, urine, distilled wa-

ter; 50:35:15)

n.d. 2 25 - 62

Varel & Parker et al. 2012 odor, pH, VFA, aromatic fermentation products,

methane, coliforms

2 L Erlenmeyer flasks with rubber stopper

600 mL (1:1 seed slurry and fresh ma-

nure)

establishing “seed slurry” over 2 - 5

months for stabiliza-tion of ph, methane and VFA-production

2 22, 38, 55

- 25, 28

Winckler & Grafe 2001 T, transformation 500 L tanks 295 L not given 4 8 - 48

Zheng & Bradford et al.

2012 T, transformation 250 mL glass bottles with teflon-lined screw caps, glovebox, Na2S,

N2

< 250 mL 1 d preconditioning 3 35 (- 277) 52

Zheng & Machewski et al.

2013 T, transformation 250 mL glass bottles with teflon-lined screw caps, glovebox, Na2S,

N2

< 250 mL 1 d preconditioning 3 15, 25, 35, 45

- 65


39

4.10 Source of manure

Although all the publications work with liquid manure, there are different approaches on application of the test substance to manure for transformation studies. First of all with VMPs contaminated manure can be generated by taking manure from previously medicated animals. The advantage of this proce-dure is the occurrence of metabolites of the parent compound. By this a more realistic transformation scenario can be studied. The deacetylation of the metabolite N-acetyl-sulfadiazine in manure after ex-cretion back to the parent compound sulfadiazine is a well-studied example. There might be many further not yet studied examples like this. (Heuer et al. 2008, Lamshöft et al. 2010) Further, VMPs influence intestinal microbiology and by this its own transformation fate in manure. Possibly intestinal built NER might also have an influence on VMP transformation in manure. This applies also to excipi-ents of the medicinal products. Considering analytical method development, taking medicated manure makes it difficult to impossible to determine recovery rates of the analytes out of the excreted and then aged manure. At this point only radioactive methods can provide a valid survey on parent compound excretion and distribution. In literature only Heuer & Spiteller et al. (2008) and Lamshöft & Spiteller et al. (2010) worked with radioactive labeled VMPs and medicated manure (14C-Sulfadiazine, 14C-Diflox-acin). Overall, 10 out of 34 studies were conducted with medicated manure. This approach seems to be more appropriate to study metabolism in the animal than transformation of compounds in manure under the regulatory perspective.

Spiking manure in laboratory scale is a much more reproducible way of generating contaminated ma-nure and the only way to handle transformation studies of biocides. By this approach it is possible to determine recovery rates with unlabeled compounds and to study sorption processes.

Most of the studies considered manure from pigs (25 out of 34), the remaining studies investigated manure from cattle. Within one study a synthetic substrate mixture including volatile fatty acids, glu-cose and starch was used (Cetecioglu & Ince et al. 2013).

Generally liquid manure is characterized as an anaerobic liquid medium. Taking uncontaminated liq-uid manure directly from a tank at a farm is the most reliable source of liquid manure. By this an adapted and realistic anaerobic and methanogenic microbial community can be assumed. 9 out of 34 studies worked with liquid manure taken out of a bigger tank at a farm. In contrast to this 16 publica-tions reported a procedure of mixing more or less fresh excrements with water and in some cases with an inoculum to produce liquid manure on laboratory scale. Out of these, only Varel & Parker et al. (2012) described a well-documented procedure of generating a “seed manure” over 2 to 5 months to mix it later with fresh manure in order to preserve a reproducible artificial liquid manure.

4 Studies worked with lagoon water, which mainly differs from liquid manure in its lower dry matter content of in these cases 1.2 - 3.6 %. Additionally, Li & Katterhenry et al. (2011) used “recycled water derived from a beef farm“. Within one publication lagoon sediment was mixed with water down to a dry matter content of 2.7 % (Ali and Hernandez 2013).

Cetecioglu & Ince et al. (2013) and Angenent & Raskin et al. (2008) took manure for transformation experiments out of a continuously running anaerobic sequencing batch reactor (ASBR), whereas Mohring & Hamscher et al. (2009) took manure directly out of a biogas plant.


40

4.11 Experimental setup

Considering the practical setup of transformation studies in literature, the whole variety of approaches becomes obvious. The amount of manure used for one replicate ranges from 1 mL (Angenent & Raskin et al. 2008) up to 295 L (Winckler & Grafe 2001). By far most of the studies were conducted with 50 - 500 mL manure. 8 studies do not report a clearly defined amount of manure used. Most studies seem to work without agitation of manure during experiments or they do not clearly report it. There are only a few studies who mention the periodically stirring of the test manure or at least directly before sam-pling the manure.

Some studies refer to several guidelines. Loke & Tjørnelund et al. (2000) and (2003) refer to ISO 11734 (ISO 1998). Mohring & Hamscher et al. (2009) refer to German VDI 4630 (VDI 2006) guideline and to DIN 38414 part 8 (DIN 1985). Szatmári & Borbély et al. (2011) refer to the former draft of the EMA-Guideline on determining the fate of veterinary medicinal products in manure (EMA 2011, VICH 2010).

As already discussed under chapter 4.3 (Aerobic vs. anaerobic conditions) many of the studies try to establish anaerobic conditions by using an inert gas for flushing headspace or solutions of the ex-periments. In general it can be differenciated between flow-through systems and batch systems (also called static or semi static systems) on this topic. There is only one publication reporting a real flow-through system (Ali and Hernandez 2013). Especially for the reported batch systems it is often not well described on how exactly produced biogas was driven out of the system or how it was dealt with the generated biogas overpressure. This is important for studies monitoring biogas production or for those studies working with 14C and monitoring mineralization.

Subsequently all the studies are shortly presented. There are two publications working with an anaer-obic sequencing batch reactor (ASBR) and one study monitoring transformation under outdoor real farm conditions.

4.11.1 N2 flow-through system

Ali and Hernandez (2013) worked with a continuous flow through of N2 and O2 in a defined ratio to reach a resulting Eh between -100 and +350 mV. With the addition of HCl or NaOH the pH was ad-justed.


41

Figure 5 Schematic diagram of microcosm system used for controlling redox potential and pH in Ali and Hernandez (2013)

4.11.2 Batch assays (no N2 flow-through)

Álvarez & Omil et al. (2010) worked with a batch assay approach. They used an inoculum to guarantee reproducible microbial starting conditions. Before starting the batch experiment N2 was flushed through the 500 mL flasks. Further Na2S was added at the beginning of the test to reach anaerobic conditions.

Arikan (2008) used a 1 L batch laboratory digester. It was filled with 800 mL fresh manure (diluted with water) from medicated pigs and 200 mL of an inoculum from the effluent of a dairy manure di-gester. After this the headspace of the digester was flushed with N2. The digesters were stirred contin-uously during the experiments. Very similar to this Arikan & Foster et al. (2006) used the same setup. Additionally, biogas production was measured by using a water displacement technique.

Blackwell & Noble et al. (2005) worked with a closed glass bottle without agitation of the manure (200 mL). It is assumed that Heuer & Spiteller et al. (2008) also worked with a batch system. Manure was stored at 20 °C in the dark. Unfortunately the used amount and the storage setup is not given.

Höltge & Kreuzig (2007), Kreuzig & Höltge (2005), Kreuzig & Höltge et al. (2007) and Kreuzig (2010) used a stoppered 300 mL test setup with 50 g mixed excrements from untreated animals. 14C-labelled biocides and pharmaceuticals were spiked to the manure and the whole setup was once flushed with nitrogen before starting the experiment. 14CO2 was trapped within a KOH-solution. The manure was not stirred. Partially redox potentials and O2-concentrations were measured to evaluate anaerobic con-ditions. Lamshöft & Spiteller et al. (2010) used a similar approach.

Kolz & Douglass et al. (2005) used amber glass vials with teflon-lined caps and filled the headspace with helium before storage. Kuchta & Cessna (2008) worked with the relatively high amount of 15.5 L manure for each replicate. It was stored in a 20-L stainless-steel container with clipdown cover to achieve similar storage conditions as they are given during storage in lagoons under a plastic cover.


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Kühne & Agthe et al. (2000) used a closed vacuum desiccator to store 1 L liquid manure under un-vented conditions. There is no mention of using N2 or another inert gas to guarantee anaerobic condi-tions.

Figure 6 Setup for transformation studies presented by Kühne & Agthe et al. (2000)

Li & Katterhenry et al. (2011) used amber bottles (250 mL) with teflon lined caps as reactors. They did not report the usage of an inert gas.

Loke & Tjørnelund et al. (2000) and Loke & Tjørnelund et al. (2003) used a 1 L batch system according to ISO 11734 (1998). 50 g manure were mixed with 525.0 ml mineral medium and 100.0 ml stock solution. Titanium(III)citrate was added as reducing agent and resazurin (1.0 mg/L) was added as a redox indicator. Test conditions were assumed to be strictly anaerobic, because no reddish coloring from the resazurin occurred and methane gas was produced constantly. To further guarantee anaero-bic conditions during the hole setup of the batch system and during sampling, it was worked under N2-gas.

Mitchell & Frear et al. (2013) mixed manure with a total solid (TS) content of 20.1 % and an inoculum with a total solids content of 1.6 %. The inoculum of primary digested sludge was obtained from a wastewater treatment plant anaerobic digester. They mainly measured the inhibition of biogas-pro-duction out of a 200 mL batch system every day for the first 7 d and then every few days for 33 d using a syringe methodology.

Mohring & Hamscher et al. (2009) worked with a commercially available anaerobic 5L fermentor (Big-atec, Rheinberg, Germany). Based on the German VDI 4630 (VDI 2006) guideline and according to DIN 38414 part 8 (DIN 1985) they studied biogas production of a liquid manure taken from a biogas plant.


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To achieve a dry matter content below 10 % they mixed the manure 1:1 with water. An inoculum of the biogas plant was added. The eight studied antibiotics were added at once to the two fermenters to realize similar degradation conditions for all of them.

Schlüsener & Bester et al. (2006) worked with 300 mL Erlenmeyer flasks closed with a fermenting tube “to maintain anaerobic conditions“. Unfortunately no information is provided on how anaerobic con-ditions were established.

Shelver & Varel (2012) report the usage of incubators/ 2 L- digester flasks. It is not mentioned how they guaranteed anaerobic conditions.

Shi & Liang et al. (2011) took 100 g dried manure, mixed it with 100 mL inoculum obtained from an anaerobic digester on a farm and filled the mixture up to 1000 mL with water. Transformation experi-ments were carried out with the digester-setup shown in Figure 7. The produced biogas was led through a NaOH-collector to absorb carbon dioxide and the equivalent volume of the not absorbed amount of remaining biogas (methane) was pushed to the collector bottle. The system was flushed with N2 before starting the transformation study and manure was stirred twice a day during the study

Figure 7 Setup for transformation studies presented by Shi and Liang et al. (2011)

Stone & Spellmann et al. (2009) filled 50 g manure into 120 mL batch reactors and purged the head-space with N2 before starting the experiments. During the whole transformation studies, they continu-ously measured numerous parameters, such as: CH4 and CO2 production, volatile fatty acids (VFA), pH, alkalinity, chemical oxygen demand (COD), VSS, acetate, propionate, butyrate, isobutyrate, and the relative abundance of hydrogenotrophic methanogens and aceticlastic methanogens.

Szatmári & Borbély et al. (2011) conducted their studies referring to the former draft of the EMA-Guid-line on determining the fate of veterinary medicinal products in manure (EMA 2011, VICH 2010). Nev-ertheless there is no mention of measured pH-values or working under N2-gas within the 300 mL bot-tles as they are used in closed bottle tests. They compared transformation under anaerobic laboratory conditions with a simultaneously conducted field study under aerobic conditions.


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Compared to all the other studies, Varel (2002) worked with a completely different approach and phi-losophy. He added the biocides Carvacrol or Thymol to the mixed excrements in order to reduce micro-bial activity and to reduce “offensive odor emissions”. In contrast to this, all the other studies were conducted in order to measure transformation under realistic conditions to assess potential environ-mental risks in the end. By intentionally adding biocides to manure, biotransformation of most VMPs will be reduced to a minimum.

Varel & Parker et al. (2012) spent a lot of efforts on establishing a well-defined seed-slurry over 2-5 months with a defined pH, methane and VFA-production. This seed slurry was later mixed with fresh manure from medicated animals. During their studies with a batch system under N2-gas they moni-tored numerous concentrations of volatile fatty acids (VFA) and aromatic fermentation products (l-lactate, acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate, phenol, p-cresol, indole and skatole) besides the parameters pH and methane gas production and others. They further reported the acclimatization of the microbial activity to the presence of monensin.

Winckler & Grafe (2001) were one of the first to study transformation of pharmaceuticals in liquid ma-nure. They worked with very large 500 l tanks under outdoor and under temperature controlled condi-tions.

Zheng & Bradford et al. (2012) studied the transformation of hormones - not a VMP - in lagoon water. Due to the limited amount of transformation studies in general and especially with lagoon water, this publication is considered within this literature review. They worked with 250 mL glass bottles and used Na2S, N2-gas and the glovebox technique to guarantee anaerobic conditions. Zheng & Machewski et al. (2013) used the same setup to study transformation of 17 α-estradiol-3-sulfate.

4.11.3 Anaerobic sequencing batch reactor (ASBR)

Within the study of Angenent & Raskin et al. (2008) a 5 L ASBR was run by sequencing through a feed step (1 min), a react step (23.2 h), a settling step (45 min), and a decant step (2–5 min). Intermittent mixing was performed by biogas recycling (1 min of biogas recycling every hour at a flow rate of 26 L/h). Tylosin half-life experiments were conducted by taking manure from the ASBR, giving it into capped 5 mL glass serum vials (prepurged with N2) and spiking it with tylosin. The vials were stored for 48 h at 25 °C in a water bath.

Cetecioglu & Ince et al. (2013) also used an ASBR with a 24-hour cycle to measure the impact of tetra-cycline on biogas production and biodegradation of a synthetic organic substrate. In contrast to An-genent & Raskin et al. (2008) they monitored the tetracycline mass balance between the influent and the effluent of the ASBR considering the sludge inside the ASBR. With this setup it was not possible to determine DT50-values.


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4.11.4 Realistic outdoor conditions

Grote & Freitag et al. (2004) conducted the only transformation study with a realistic outdoor scenario with Chlortetracycline (CTC), Sulfadiazine (SDZ) and Trimethoprim (TMP). The medicated pigs ex-creted the pharmaceuticals over a long period of time. By this all the relevant metabolites were consid-ered. Unless it is not easily possible to determine DT50-values with this approach, but it is possible to study the realistic transformation of a pharmaceutical.

4.12 Parameters

Within all the publications the following physical, chemical and biological parameters were measured or controlled during transformation studies:

Redoxpotential Eh [mV], dry matter content [%], pH, dissolved O2-content [mg/kg], NH4-N [g/kg], Ntotal [g/kg], total organic carbon (TOC) [g/kg], total carbon [g/kg], biological oxygen demand (BOD) [g/kg], chemical oxygen demand (COD) [g/kg], temperature [°C], volatile suspended solids (VSS), phosphorus [g/kg], conductivity [µs/cm], Cl-, Br-, NO3-, Na, K, Ca, Fe, Mg, Al, Si, Cu, Zn, relative abundance of hy-drogenotrophic methanogens and aceticlastic methanogens. volatile fatty acids (VFA) and aromatic fermentation products (l-lactate, acetate, propionate, isobutyrate, butyrate, isovalerate, valerate, isocaproate, caproate, phenol, p-cresol, indole and skatole), methane/biogas production, mineraliza-tion [%], 50 and 90 % disappearance time (DT50, DT90), transformation products (TP), non-extracta-ble residues (NER), mass balance/recovery [%], liquid-solid distribution.


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4.13 Conclusions

The available studies on transformation of biocides and veterinary medicinal products in manure show large variations in the experimental set up and conditions such as temperature, redox potential, matrix effects, and physico-chemical properties.

Most frequently investigated VMPs are from the class of antibiotics, namely sulfonamides, tetracy-clines, and macrolid antibiotics. In 10 out of the 34 studies, excrements or manure from medicated livestock was considered, all other studies are based on spiked manure. Most of the studies considered manure from pigs (25 out of 34), the remaining studies investigated manure from cattle or in one case used synthetic manure. Large differences are represented in terms of study duration and temperature, ranging from 2 to 374 days and 5 °C to 55 °C, respectively. Many studies suffer from insufficient control of basic parameters. Only six publications give information on the redox potential of the manure used for transformation. Further, the characterization of the matrix in many cases is inadequate due to miss-ing parameters such as dry matter content, pH, and TOC. Information on dissipation rates or half-lives, transformation products, generation of methane and non-extractable residues (NER) are not available in the majority of the considered publications.

Overall, it can be stated that the majority of the studies describes at least one fundamental parameter of the experimental conditions poorly. Considering all the different approaches one can conclude that it is inevitable to give specific guidance for studies on transformation in manure in general and espe-cially with regard to the applicability and acceptability of studies in regulatory contexts. Knowing that all the parameters - as they are studied particularly within single publications - do affect the basic out-come of a transformation study, it is necessary to standardize them or at least report them. Parameters as temperature, dry matter content, origin and preconditioning of the manure, pH and Eh do have massive effects on transformation rates or routes of VMPs and biocides in liquid manure. All these pa-rameters are relatively easy to measure and should be monitored - or better standardized where possi-ble- mandatorily. Another topic with knowledge gaps is related to the composition, the development or spread of resistance, or adaption and activity of the microbial community. For future studies this topic always needs to be addressed.

There are different tasks of academic interest within the research on monitoring data and transfor-mation studies in manure, which are not directly related to the main question of how to standardize experiments on a laboratory scale to assess the environmental exposure by VMPs and biocides.

As an outcome of the summarized monitoring studies, a widespread distribution of VMPs in manure can be concluded. We found only one transformation study at real manure storage tanks. This is an important area for research to study transformation under realistic conditions. Comparing such realis-tic outdoor results with those produced on laboratory scale is important to validate the outcome of laboratory studies and to evaluate different experimental setups.


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